Our preliminary studies, which include immuno-, chemical, and enzymatic assays, Edman sequencing, matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), and proton nuclear magnetic resonance spectroscopy ('H-NMR) indicate that E. coli protein OmpA is modified by cPHB, an amphiphilic, salt-solvating molecule. OmpA is a major outer membrane protein that has served as a paradigm for studies of sorting and assembly of proteins of Gram-negative bacteria into the outer membrane, but despite extensive investigations these mechanisms are still not understood. Previous studies by other investigators have determined that the sorting signal is contained within the eighth B membrane strand. Our preliminary studies indicate that the binding site for cPHB is a tyrosine residue in the eighth f3-strand. Site-directed mutagenesis of this residue produced cPIIB-minus OmpA. OmpA is also widely regarded as a porin, but the question of whether it is capable of forming open pores remains controversial. Liposome-swelling assays and single-channel planar bilayer studies indicate a small percentage of molecules have open pores, whereas high-resolution X-ray structures show no evidence of a continuous water channel through the protein. Our hypothesis is that cPHB-modification is implicated in the sorting and proper membrane assembly and/or pore formation by OmpA. Here we propose to investigate the effects of cPHB on OmpA structure and function. The ability of wild type and cPHB-minus OmpA proteins to incorporate and assembly properly in the outer membrane will be examined in studies of heat-modifiability, sensitivity to proteases, and sensitivity to OmpA-specific phages. The capacity of wild type and cPFIBminus OmpA molecules to form open pores in the membrane will be investigated in single-channel planar bilayer studies. Finally, enzyme(s) responsible for cPHB synthesis in E. coli will be identified.